Abstract: Eastern, Venezuelan, and Western equine encephalitis (EEE, VEE, WEE) viruses (Alphavirus; Togaviridae) are mosquito-borne viruses causing severe encephalitis in humans and horses. All three viruses are listed as category B agents by both NIAID and CDC and as potential bioterrorism/biowarfare (BT/BW) agents. There are enormous knowledge gaps concerning transmission potential, mosquito-host determinants, pathogenesis from aerosolized virus, acquired and innate immune responses, etc. for this group of viruses, especially EEEV and WEEV. In this project, we will develop alphavirus (EEEV and WEEV) infectious cDNA clones to determine molecular mechanisms of viral replication and pathogenesis in mice and infectivity of mosquitoes that may serve as vectors in secondary transmission during a BT/BW event. We will also develop stable, double subgenomic EEEV, VEEV, and WEEV transducing viruses to characterize their ability to replicate and express a reporter gene in infected mice and vectors. We will also conduct pathogenesis studies focusing primarily on EEEV and WEEV in small animal models, but will also include transducing viruses from the three alphaviruses in these studies. We will focus on pathogenesis in mice following infection by vector and aerosol routes of infection. Finally we will characterize virus-vector interactions for all three viruses that may lead to increased transmission potential by vectors of BT/BW agents and emerging alphavirus pathogens. Project Interactions: The Pi's of the project are Drs. Ann M. Powers and Ken E. Olson. Drs. Powers and Olson are located at the Centers for Disease Control, Division of Vector-borne Infectious Diseases and Arthropod-borne Infectious Diseases Laboratory (CSU) in Fort Collins, Co., respectively. Dr. Powers and Olson both have considerable experience in alphavirus biology, molecular biology, vector biology and are developing expertise in alphavirus interactions with small animal models. In addition, the project will interact with other investigators in the RCE. Specifically, with Drs. Kedl, Blair, and Dow on Lanacs studies (II.C.6), Drs. Titus and Beaty (II.B.2.) and Dr. Chang (II.B.3) on novel vaccine technologies, Dr. Morrey (II.G) in sharing infectious alphavirus clones and transducing viruses developed in this project and Dr. Fujinami and Foy (II.H.2) on RNAi-based antiviral therapies. This project will be a resource for developing protocols, viruses, and reagents that can be used by other members of the RCE. Finally, this project will be dependent on two SFC's: Animal Models Core (III.A) and Genomics/Proteomics Core (III.C).